The Mechanism Of BmNPV LEF-2 Regulating The Cell Cycle Of Silkworm

The virus has a simple structure and needs completes its own proliferation only with the help of various functions of the host cell.Most viruses can complete their life activities by regulating the host cell cycle to create an internal environment that is conducive to virus proliferation.Baculovirus is the earliest discovered and most studied insect virus,among which Bombyx mori nucleopolyhedrovirus（BmNPV）is the representative species of baculovirus.BmNPV is the pathogen of blood sepsis in sericulture,which brings huge economic loss to sericulture production every year.Our previous study found that the cell cycle of BmNPV infected BmN-SWU1 cells was blocked in G2/M phase to facilitate viral proliferation and replication,but the specific regulatory mechanism needs to be further analyzed.Therefore,this study takes BmNPV as the research object,identifies the key viral genes that the virus regulates the host cell cycle and improves the interaction network between baculovirus and host cells,which can provide theoretical support for the prevention and control of BmNPV.In view of this,this study analyzed the basic characteristics and functions of BmNPV late expression factor 2（lef-2）,identified the effect of BmNPV lef-2 gene on the host cell cycle,explained the mechanism of BmNPV lef-2 gene inducing host DNA damage response（DDR）to regulate cell cycle and further improved the research on the mechanism of baculovirus regulating host cell cycle.The main results of this study are as follows:1.The analysis of basic characteristics and function of BmNPV lef-2To analyze the functional characteristics of BmNPV LEF-2,the protein sequence homology alignment and phylogenetic tree analysis of BmNPV LEF-2 were carried out.It was found that the protein sequence of BmNPV LEF-2 was relatively conservative among baculoviruses and Alpha baculovirus group I was clustered into one branch,with a close evolutionary relationship.The expression of BmNPV lef-2 gene in BmN-SWU1cells infected with BmNPV was detected by RT-PCR and Western blotting.The results showed that the transcript of BmNPV lef-2 gene was detected at 6 hours post infection（h p.i.）and BmNPV LEF-2 protein was detected at 12 h p.i.,and the expression levels increased gradually with virus proliferation.Subcellular localization analysis of BmNPV LEF-2 showed that BmNPV LEF-2 was located in the nucleus.Furthermore,immunofluorescence was used to explore the co-localization of BmNPV LEF-2 protein and IE-1.The results showed that they were co-located in the nucleus,indicating that BmNPV LEF-2 protein was located in the virogenic stroma.In order to further explore the effect of BmNPV lef-2 gene on virus proliferation,BmNPV lef-2 gene deficient recombinant virus and revertant recombinant virus were constructed.It was found that the virus could not proliferate after deletion of BmNPV lef-2 gene,indicating that BmNPV lef-2 gene is the key gene of virus proliferation and replication.2.The regulation of BmNPV lef-2 on host cell cycleTo identify the effect of BmNPV lef-2 gene on cell cycle,EdU labeling test and flow cytometry analysis were used to dected DNA replication.The result of EdU labeling showed that overexpression of BmNPV lef-2 gene inhibited host DNA replication,and flow cytometry analysis showed that overexpression of BmNPV lef-2 gene blocked the host cell cycle in G2/M phase.In order to further explore the mechanism of BmNPV lef-2 blocking the G2/M phase of cell cycle,the transcription level and protein level of cyclin B and cyclin dependent kinase I（CDK1）was analyzed,which was the key regulatory genes of G2/M phase.qRT-PCR results showed that the transcription levels of BmCyclin B and BmCDK1 were significantly downregulated after overexpression of BmNPV lef-2.Western blotting results showed that the expression of BmCyclin B and BmCDK1 protein were significantly downregulated after overexpression of BmNPV lef-2.The above results showed that BmNPV lef-2 downregulated the expression of BmCyclin B and BmCDK1,and blocked the cell cycle in G2/M phase.3.The effect of vBm^milef2 recombinant virus on host cell cyclesIn order to analyze whether BmNPV lef-2 gene is the key gene affecting cell cycle arrest,this study explored the effect of BmNPV on host cell cycle after interfering with BmNPV lef-2 gene.Firstly,we constructed BmNPV lef-2 gene interference recombinant virus vBm^milef2,then,EdU labeling test and flow cytometry analyse were used to detect the effect of vBm^milef2 on cell cycle.EdU labeling results showed that interference with BmNPV lef-2 gene would weaken the effect of BmNPV on host cell DNA replication compared with vBm^EGFP.Flow cytometry analysis showed the same experimental results as EdU labeling test.qRT-PCR and Western blotting were further used to detect the effect of vBm^milef2 on the expression of key regulatory genes BmCyclin B and BmCDK1 in G2/M phase.qRT-PCR showed that compared with vBm^EGFP,the expression of BmCyclin B and BmCDK1 were significantly up-regulated at 12 h,24 h and 48 h,which indicated that interfering with BmNPV lef-2 gene would weaken the effect of BmNPV on the transcription level of key genes in G2/M phase of host cell cycle;Western blotting showed that compared with vBm^EGFP,the expression of BmCyclin B and BmCDK1 protein increased,which was consistent with the qRT-PCR results.The above results showed that interference with BmNPV lef-2 will weaken the effect of BmNPV on host cells,which indicating that BmNPV lef-2 gene is an important viral gene for BmNPV to regulate cell cycle.To further explore the mechanism of BmNPV lef-2 gene regulating cell cycle,immunofluorescence and co-immunocoprecipitation were used to detect whether BmNPV LEF-2 interacted directly with G2/M phase regulating genes to regulate cell cycle.The results showed that there was no direct interaction between BmNPV LEF-2and BmCyclin B and BmCDK1.4.The study on the mechanism of BmNPV lef-2 regulating host cell cycle through DNA damage responseIt has been found that DNA damage response can directly act on the key cyclins regulating cell cycle transition and then participate in the regulation of cell cycle.In order to study the mechanism of BmNPV lef-2 gene regulating host cell cycle arrest in G2/M phase,the effect of BmNPV on DDR was detected.The results of Western blotting showed that the expression of P53 protein increased after virus infection,indicating that BmNPV can induce DDR and the degree of induction of DDR was positively correlated with the dose of virus infection.Then,qPCR and Western blotting experiments were used to detect the effect of DDR on virus proliferation.qPCR results showed that the expression of virus gp41 increased after DDR induction and decreased after DDR inhibition;Western blotting showed that the expression of VP39 protein increased after inducing DDR and decreased after inhibiting DDR,indicating that DDR is conducive to virus proliferation and replication.Furthermore,in order to detect the effect of DDR on cell cycle,flow cytometry was used to analyze the cell cycle changes.It was found that the cells in G2/M phase increased significantly after adding DDR inducer,indicating that DDR can induce cell cycle arrest in G2/M phase.Then we explored the effect of BmNPV lef-2 gene on DDR.Western blotting showed that after overexpression of BmNPV lef-2gene,the expression of P53 protein increased,indicating that BmNPV LEF-2 could induce DDR and qPCR showed that the expression of gp41 increased after overexpression of BmNPV lef-2 gene,indicating that BmNPV LEF-2 was conducive to virus proliferation and replication.In order to further verify whether BmNPV lef-2 gene regulates the cell cycle through DDR,DDR inhibitor and inducer were added respectively under the condition of overexpression of BmNPV lef-2 gene and the changes of cell cycle were analyzed by flow cytometry.The results showed that compared with the control,the cells blocked in G2/M phase were significantly reduced by inhibiting DDR after overexpression of BmNPV lef-2 gene,while the cells blocked in G2/M phase were significantly increased by inducing DDR after overexpression of BmNPV lef-2 gene.These results suggest that BmNPV lef-2 gene can regulate the host cell cycle by inducing DDR.In conclusion,this study identified that BmNPV lef-2 downregulated the expression of key regulatory genes in G2/M phase by regulating DNA damage response,so as to regulate the host cell cycle.